Abstract
Objective: To make in vitro maturation a successful treatment, it is essential to achieve a more profound insight into the function of the oocyte. It has been shown that active induction of meiotic resumption by FSH in mouse cumulus-enclosed oocytes (CEO) depends on phosphoinositide metabolism and intracellular calcium. However, it is not known whether these pathways play a role in cumulus cells or the oocyte. This study was conducted to assess whether in denuded oocytes phosphoinositide metabolism and intracellular calcium are also involved in the reinitiation of meiosis promoted by FF-MAS, a naturally occurring sterol synthesized by follicle cells. Design: CEO and naked oocytes (NkO), cultured under conditions which prevent spontaneous meiotic resumption, were exposed to FSH or FF-MAS, respectively, in the presence or absence of phosphoinositide metabolism inhibitors (neomycin and LiCl) or an intracellular calcium chelator (BAPTA/AM). Materials and Methods: Oocytes were collected from large antral follicles of three-week-old female mice stimulated with 5 IU of pregnant mare’s serum gonadotropin, and were cultured in α-MEM medium supplemented with 4 mM hypoxanthine, to prevent spontaneous germinal vesicle breakdown (GVBD). CEO were stimulated with FSH (0.1 IU.ml−1) whereas NkO were stimulated with FF-MAS (10 μM). A proportion of oocytes was also exposed to the continued presence of either neomycin (1 mM) or LiCl (10 mM), or loaded with BAPTA/AM (15 μM). After 20 h, the rate of GVBD was assessed by visual examination and compared between the different groups. Results: Oocytes cultured in the presence of hypoxanthine alone were unable to resume meiosis. As expected, FSH and FF-MAS were able to induce meiotic resumption in CEO and NkO, respectively (81.1% and 84.2% GVBD). Neomycin and LiCl significantly inhibited FSH-mediated GVBD in CEO (12.9% and 15.2% GVBD, respectively). Similarly, loading oocytes with BAPTA/AM repressed meiotic resumption promoted by FSH (29.7% GVBD). However, in NkO stimulated with FF-MAS, the two phosphoinositide inhibitors as well as the calcium chelator failed to prevent meiotic resumption, GVBD occurring at a high rate, comparable to the control group (82.8%, 91.2% and 81.9%, respectively). Conclusions: In CEO arrested at the GV stage, FSH-induced meiotic resumption requires activation of phosphoinositide metabolism and release of intracellular calcium. However FF-MAS, which is thought to be synthesized by cumulus cells following acute FSH stimulation, can promote GVBD in NkO irrespective of the presence of neomycin, LiCl and BAPTA/AM. This suggests that, during meiotic resumption, these signaling pathways are involved in the response of cumulus cells to FSH. On the contrary, they do not appear to play a role in the mechanism which regulates the reinitiation of the meiotic cycle within the oocyte.
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
